Vancomycin (VAN) is among those antibiotics for which therapeutic drug monitoring is highly recommended. For this purpose a reliable method with small sample volume was required for quantification of VAN in human plasma. Therefore, a selective and sensitive method of high performance liquid chromatography was developed and validated. The separation was carried out isocratically by using a mobile phase NH4H2PO4 (50 mM, pH 2.2)–acetonitrile (88:12, v/v) at a flow rate of 0.36 mL/min on a nucleodur C18 column (125 mm × 4.6 mm, 5 µm) with UV detection at 205 nm. Sample preparation was done by deproteination of plasma with 70 % perchloric acid and a liquid/liquid extraction. Validation was performed according to the European Medicines Agency guideline. The method showed linearity over the range of 0.25–60 mg/L with a coefficient of determination r2 ≥ 0.999 and a lower limit of quantification of 0.25 mg/L. No interference was observed in blank plasma samples at the retention time of VAN. The percentage relative recovery and coefficient of variation (CV%) values for accuracy and precision were within the acceptable limits. Stability was proved at room temperature for 24 h, after repeated freeze and thaw cycles and storage at −20 °C for 3 months. A good correlation was observed (r = 0.947) by comparing with the results of an immunoassay (PETINIA, Siemens) in 289 samples. In conclusion the method proved simple, sensitive and cost effective for quantification of VAN in human plasma.